All vertebrates develop limbs by similar mechanisms, but only one group, urodele amphibians (salamanders and newts) have the remarkable ability to regenerate their limbs. Our goal is to understand the molecular events that occur when differentiated limb cells reenter the developmental pathway to reengage in pattern formation. During limb development, flank cells must first be specified as fore- or hind limb, and establish anterior-posterior and dorsal-ventral polarity prior to generating the proximal-distal axis. In contrast, during regeneration only the proximal- distal axis has to be re-developed. In all animals, genes of the Hox clusters are involved in pattern formation, both along the main head to tail axis and within the limbs. The expression patterns and functional analyses of genes of one Hox cluster, the HoxA genes, indicate their involvement in patterning of the proximal-distal limb axis in development. Since limb regeneration is the process of reforming the proximal-distal axis, we hypothesize that these genes will have a key regulatory function in limb regeneration. For this reason, we propose to analyze the expression and function of HoxA genes in regenerating axolotl limbs. In preliminary studies, we have discovered that HoxA genes are reexpressed in the initiation phase of regeneration. Both HoxA13 and A9 are reexpressed in the limb stump as an early response to amputation, prior to formation of the blastema that gives rise to the new structures. Our data suggest that HoxA genes do not obey the rules of temporal and spatial colinearity that are the hallmark of their expression during limb development. The order in which HoxA genes are reexpressed, and how the combinatorial domains of gene expression that characterize each limb segment (the Hox code) arise, will be investigated in our experiments. We will use information from classical experiments combined with experimental manipulations of gene expression (overexpression and inhibition of expression) to investigate how events in the regeneration cascade are causally linked to HoxA gene expression. We will find out whether activation of HoxA genes is unique to regeneration by studying non- regenerating wounds. We will examine the role of the epidermis (and FGF) and nerves in the initiation and maintenance of HoxA expression. We will study the role of HoxA genes in the formation of specific limb segments along the proximal-distal axis. We will investigate the relationship between the effects of retinoic acid on the pattern of regenerating limbs and its effects on HoxA gene expression. We will study the relationship between differential adhesiveness of cells from different proximal-distal limb levels and their different HoxA codes. With an understanding of the role of HoxA genes in regeneration, we will further our understanding of why limbs of other vertebrates, including humans, are unable to initiate the regeneration cascade, thus providing insights into future strategies for therapy.